Abstract: Structural biology methods including X-ray crystallography, NMR and cryo-electron microscopy (EM) for determining the three-dimensional structures of biomolecules in vitro were critical to the rapid expansion in our molecular understandings of biological processes. However, as the challenge shifts towards the higher order architecture of macromolecular machineries, these methods all face difficulties, especially for studies in the native cellular environment. To address this challenge, we propose to develop a new approach based on light microscopy. Enabled by our recently developed super-resolution optical microscopy techniques, the key of this approach is to fluorescently label the components of the complex and determine their relative positions through single- molecule localization. To develop this method, we will further improve the resolution of super-resolution microscopy to the molecular level by optimizing the instrument design, protein labeling methods and data analysis algorithm. We will also apply this method to investigating the architecture of yeast nuclear pore complex. As a next step, through position mapping of individual domains of protein units and computation to fit atomic models into the context of molecular complexes, we will obtain full pseudo-atomic resolution structures of large complexes. The proposed research will establish a new structural determination tool that complements existing methods. It is uniquely advantageous is the ability to study large macromolecular assembly in situ without purification, owing to the noninvasive nature of optical microscopy. Thus, it will greatly help understanding the structure and structure-function relationship of a broad range of macromolecular assemblies, providing insights of how they functions in the native environment, especially for those difficult to be extracted from the cell. Public Health Relevance: Understanding the structure of a biomolecule or a macromolecular complex, just like knowing the blueprint of a building, lays the foundation of insights to its function mechanism and subsequent drug development. The proposed research is aimed at establishing a new structural biology method based on super-resolution optical microscopy to determine the molecular architecture of macromolecular complexes in the native environment. It will greatly widen the spectrum of cellular components that can be structurally characterized, thus strengthening our ability for the mechanistic understanding of biomedical problems.